The goal of the proposed experiments is to determine the mechanism of tissue-specific regulation of tRNA transcription. We will focus on two tRNA/A1a genes of silkworms, one of which (C) is transcribed constitutively, and the other (SG) only in silkglands. We will use both in vitro and in vivo assays of promoter function, and will analyze in detail protein-DNA complexes that include the upstream segment of C and SG promoters. This segment is interesting because it controls differential transcription from the two promoters in vitro, in homologous transcription systems. The upstream promoters for both genes are contained within -35 bp DNA segments immediately upstream of their transcription initiation sites, and promoter function for the C gene is conferred by two AT-rich boxes within this segment. The specific sequence or structural distinction that is responsible for differential transcription from C and SG promoters is not known. We plan the following experiments to understand how this critical promoter segment functions: We will use both in vitro and in vivo assays (in cultured cells and in intact silkglands) to identify the sequence and/or structural features that functionally distinguish the upstream promoters of C and SG genes, and that confer silkgland specificity on the SG promoter. We will compare the interaction of TFIIIB with C and SG upstream promoters, using TFIIIB isolated either from silkglands or from a tissue (ovaries) in which the SG promoter is inactive. Based on this analysis, we will identify and purify candidate polypeptides that are likely to be key discriminators between C and SG promoters. Polypeptides that are found to regulate SG transcription in vitro will be functionally tested in vivo by determining their ability to regulate SG transcription when ectopically expressed in cultured Drosophila and silkworm cells and in intact, differentiated silkworm tissues.